Alcohol-induced decrease in muscle protein synthesis associated with increased binding of mTOR and raptor: Comparable effects in young and mature rats.

Lang CH, Pruznak AM, Nystrom GJ, Vary TC - Nutr Metab (Lond) (2009)

Bottom Line:
Acute alcohol (EtOH) intoxication decreases muscle protein synthesis via inhibition of mTOR-dependent translation initiation.Furthermore, some in vivo-produced effects of EtOH vary in an age-dependent manner.The EtOH-induced changes in both groups were associated with a concomitant reduction in 4E-BP1 phosphorylation, and redistribution of eIF4E between the active eIF4E.eIF4G and inactive eIF4E.4EBP1 complex.

Affiliation: Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA. clang@psu.edu.

ABSTRACT

Background: Acute alcohol (EtOH) intoxication decreases muscle protein synthesis via inhibition of mTOR-dependent translation initiation. However, these studies have been performed in relatively young rapidly growing rats in which muscle protein accretion is more sensitive to growth factor and nutrient stimulation. Furthermore, some in vivo-produced effects of EtOH vary in an age-dependent manner. The hypothesis tested in the present study was that young rats will show a more pronounced decrement in muscle protein synthesis than older mature rats in response to acute EtOH intoxication.

Methods: Male F344 rats were studied at approximately 3 (young) or 12 (mature) months of age. Young rats were injected intraperitoneally with 75 mmol/kg of EtOH, and mature rats injected with either 75 or 90 mmol/kg EtOH. Time-matched saline-injected control rats were included for both age groups. Gastrocnemius protein synthesis and the activity of the mTOR pathway were assessed 2.5 h after EtOH using [³H]-labeled phenylalanine and the phosphorylation of various protein factors known to regulate peptide-chain initiation.

Results: Blood alcohol levels (BALs) were lower in mature rats compared to young rats after administration of 75 mmol/kg EtOH (154 ± 23 vs 265 ± 24 mg/dL). However, injection of 90 mmol/kg EtOH in mature rats produced BALs comparable to that of young rats (281 ± 33 mg/dL). EtOH decreased muscle protein synthesis similarly in both young and high-dose EtOH-treated mature rats. The EtOH-induced changes in both groups were associated with a concomitant reduction in 4E-BP1 phosphorylation, and redistribution of eIF4E between the active eIF4E.eIF4G and inactive eIF4E.4EBP1 complex. Moreover, EtOH increased the binding of mTOR with raptor in a manner which appeared to be AMPK- and TSC-independent. In contrast, although muscle protein synthesis was unchanged in mature rats given low-dose EtOH, compared to control values, the phosphorylation of rpS6 and eIF4G was decreased.

Conclusion: These data indicate that muscle protein synthesis is equally sensitive to the inhibitory effects of EtOH in young rapidly growing rats and older mature rats which are growing more slowly, but that mature rats must be given a relatively larger dose of EtOH to achieve the same BAL. Based on the differential response in mature rats to low- and high-dose EtOH, the decreased protein synthesis was associated with a reduction in mTOR activity which was selectively mediated via a reduction in 4E-BP1 phosphorylation and an increase in mTOR.raptor formation.

Figure 3: Effect of acute alcohol intoxication on 4E-BP1 phosphorylation and the formation of the active eIF4F complex in skeletal muscle from young and mature rats. Groups are the same as described in Figure 1. Gastrocnemius was collected 2.5 h after administration of alcohol or saline (control). Insert above each graph: representative Western blots. Panels A and B: densitometric analysis of immunoblots of γ-phosphorylated 4E-BP1 and total eIF4E in muscle homogenate, respectively, Panels C and D: eIF4E was immunoprecipitated (IP) and the amount of 4EBP1 and eIF4G bound to eIF4E was assessed by immunoblotting (IB). There was no age- or alcohol-induced change in total eIF4E in the immunoprecipitate (data not shown). In panel C, the α- and β-isoforms of 4E-BP1 are indicated. Values (means ± SEM) are expressed relative to the young saline-treated control group. Sample size was 10, 10, 6, 7, 9, and 9 for the six groups, respectively. Values with different letters are significantly different from each other, P < 0.05. Values which share a common letter are not statistically different.

Mentions:
4E-BP1 is phosphorylated in a hierarchical manner and the various phosphorylated forms of the protein are resolved into three bands which are designated α-, β-, and γ [30]. In contrast to the hypo-phosphorylated α- and β-isoforms, the γ-isoform is highly phosphorylated and does not bind eIF4E. There was no difference in either the total amount of 4E-BP1 (data not shown) or the amount of γ-phosphorylated 4E-BP1 in gastrocnemius of young and mature rats (Figure 3A). Acute alcohol intoxication reduced 4E-BP1 γ-phosphorylation by 60% in young rats. A comparable decrease was seen in mature rats administered the high-dose of alcohol, but not in mature animals given the low-dose of alcohol.

Figure 3: Effect of acute alcohol intoxication on 4E-BP1 phosphorylation and the formation of the active eIF4F complex in skeletal muscle from young and mature rats. Groups are the same as described in Figure 1. Gastrocnemius was collected 2.5 h after administration of alcohol or saline (control). Insert above each graph: representative Western blots. Panels A and B: densitometric analysis of immunoblots of γ-phosphorylated 4E-BP1 and total eIF4E in muscle homogenate, respectively, Panels C and D: eIF4E was immunoprecipitated (IP) and the amount of 4EBP1 and eIF4G bound to eIF4E was assessed by immunoblotting (IB). There was no age- or alcohol-induced change in total eIF4E in the immunoprecipitate (data not shown). In panel C, the α- and β-isoforms of 4E-BP1 are indicated. Values (means ± SEM) are expressed relative to the young saline-treated control group. Sample size was 10, 10, 6, 7, 9, and 9 for the six groups, respectively. Values with different letters are significantly different from each other, P < 0.05. Values which share a common letter are not statistically different.

Mentions:
4E-BP1 is phosphorylated in a hierarchical manner and the various phosphorylated forms of the protein are resolved into three bands which are designated α-, β-, and γ [30]. In contrast to the hypo-phosphorylated α- and β-isoforms, the γ-isoform is highly phosphorylated and does not bind eIF4E. There was no difference in either the total amount of 4E-BP1 (data not shown) or the amount of γ-phosphorylated 4E-BP1 in gastrocnemius of young and mature rats (Figure 3A). Acute alcohol intoxication reduced 4E-BP1 γ-phosphorylation by 60% in young rats. A comparable decrease was seen in mature rats administered the high-dose of alcohol, but not in mature animals given the low-dose of alcohol.

Bottom Line:
Acute alcohol (EtOH) intoxication decreases muscle protein synthesis via inhibition of mTOR-dependent translation initiation.Furthermore, some in vivo-produced effects of EtOH vary in an age-dependent manner.The EtOH-induced changes in both groups were associated with a concomitant reduction in 4E-BP1 phosphorylation, and redistribution of eIF4E between the active eIF4E.eIF4G and inactive eIF4E.4EBP1 complex.

Affiliation:
Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA. clang@psu.edu.

ABSTRACT

Background: Acute alcohol (EtOH) intoxication decreases muscle protein synthesis via inhibition of mTOR-dependent translation initiation. However, these studies have been performed in relatively young rapidly growing rats in which muscle protein accretion is more sensitive to growth factor and nutrient stimulation. Furthermore, some in vivo-produced effects of EtOH vary in an age-dependent manner. The hypothesis tested in the present study was that young rats will show a more pronounced decrement in muscle protein synthesis than older mature rats in response to acute EtOH intoxication.

Methods: Male F344 rats were studied at approximately 3 (young) or 12 (mature) months of age. Young rats were injected intraperitoneally with 75 mmol/kg of EtOH, and mature rats injected with either 75 or 90 mmol/kg EtOH. Time-matched saline-injected control rats were included for both age groups. Gastrocnemius protein synthesis and the activity of the mTOR pathway were assessed 2.5 h after EtOH using [³H]-labeled phenylalanine and the phosphorylation of various protein factors known to regulate peptide-chain initiation.

Results: Blood alcohol levels (BALs) were lower in mature rats compared to young rats after administration of 75 mmol/kg EtOH (154 ± 23 vs 265 ± 24 mg/dL). However, injection of 90 mmol/kg EtOH in mature rats produced BALs comparable to that of young rats (281 ± 33 mg/dL). EtOH decreased muscle protein synthesis similarly in both young and high-dose EtOH-treated mature rats. The EtOH-induced changes in both groups were associated with a concomitant reduction in 4E-BP1 phosphorylation, and redistribution of eIF4E between the active eIF4E.eIF4G and inactive eIF4E.4EBP1 complex. Moreover, EtOH increased the binding of mTOR with raptor in a manner which appeared to be AMPK- and TSC-independent. In contrast, although muscle protein synthesis was unchanged in mature rats given low-dose EtOH, compared to control values, the phosphorylation of rpS6 and eIF4G was decreased.

Conclusion: These data indicate that muscle protein synthesis is equally sensitive to the inhibitory effects of EtOH in young rapidly growing rats and older mature rats which are growing more slowly, but that mature rats must be given a relatively larger dose of EtOH to achieve the same BAL. Based on the differential response in mature rats to low- and high-dose EtOH, the decreased protein synthesis was associated with a reduction in mTOR activity which was selectively mediated via a reduction in 4E-BP1 phosphorylation and an increase in mTOR.raptor formation.